Quantitative analysis of enteric neurons containing choline acetyltransferase and nitric oxide synthase immunoreactivities in the submucosal and myenteric plexuses of the porcine colon

The enteric nervous system (ENS) controls gastrointestinal functions. In large mammals’ intestine, it comprises an inner (ISP) and outer (OSP) submucous plexus and a myenteric plexus (MP). This study quantifies enteric neurons in the ISP, OSP, and MP of the pig ascending (AC) and descending colon (DC) using the HuC/D, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) neuronal markers in whole mount preparations with multiple labeling immunofluorescence. We established that the ISP contains the highest number of HuC/D neurons/mm2, which were more abundant in AC vs. DC, followed by OSP and MP with similar density in AC and DC. In the ISP, the density of ChAT immunoreactive (IR) neurons was very similar in AC and DC (31% and 35%), nNOS-IR neurons were less abundant in AC than DC (15% vs. 42%, P < 0.001), and ChAT/nNOS-IR neurons were 5% and 10%, respectively. In the OSP, 39–44% of neurons were ChAT-IR in AC and DC, while 45% and 38% were nNOS-IR and 10–12% were ChAT/nNOS-IR (AC vs. DC P < 0.05). In the MP, ChAT-IR neurons were 44% in AC and 54% in DC (P < 0.05), nNOS-IR neurons were 50% in both, and ChAT/nNOS-IR neurons were 12 and 18%, respectively. The ENS architecture with multilayered submucosal plexuses and the distribution of functionally distinct groups of neurons in the pig colon are similar to humans, supporting the suitability of the pig as a model and providing the platform for investigating the mechanisms underlying human colonic diseases.